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Molecular Oncology, Markers, Clinical Correlates |
Departments of Biophysics [H. L., E. K. R.] and Gynecologic Oncology [K. S., C. T.], The Norwegian Radium Hospital, 0310 Oslo 3, Norway
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Identification of the biological parameter most important for disease control can be useful for selection of patients for different treatment regimes and for development of efficient strategies to influence the control probability by changing the biological parameter. Such identification should be based on studies of several parameters in the same population of patients. Comparison of results from different studies is complicated because studies often differ with respect to number of patients, stage of disease, treatment regime, and follow-up time. In the present work, tumor oxygen tension (pO2), vascular density, cell density, and frequency of mitosis and apoptosis were measured before the start of treatment (40 patients) and after 2 weeks of radiotherapy (22 patients) in patients with carcinoma of the uterine cervix. The aim was to investigate whether one of the parameters was more important for disease control than the others. Three sets of data were considered: the pretreatment parameters, the parameters measured after 2 weeks of radiation, and the changes in the parameters during this time. The measurements after 2 weeks were performed during the early phase of radiotherapy, i.e., before significant tumor shrinkage had occurred. Knowledge of possible relationships between disease control and biological parameters of this phase would be particularly useful because strategies for selecting patients to adjuvant treatments and/or for changing the biological parameters should be initiated as early as possible during therapy. The pO2 was measured by use of polarographic needle electrodes. Vascular density, cell density, and frequency of mitosis and apoptosis were determined by histological analysis of biopsies taken after each pO2 measurement.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Radiotherapy was given as combined external irradiation and brachytherapy with curative intent to all but four patients. External irradiation was delivered with 10 MV or 16 MV photons by use of a linear accelerator. A total dose of 50 Gy in fractions of 2 Gy per day five times/week was given to the pelvic region with a four-field box technique. Endocavitary brachytherapy was delivered by use of a high dose rate 192Ir afterloading machine. A total dose of 29–34 Gy was given in seven to eight fractions to point A. Adjuvant chemotherapy was not used.
The patients were followed up with clinical examinations every third month for the first 2 years and thereafter twice a year. MR imaging of retroperitoneum and X-ray of the thorax were performed during the first follow-up examination, after 2 months, after 1 year, and thereafter when symptoms of recurrent disease were seen. Three different end points were used in evaluation of disease control: overall survival, disease-free survival, and locoregional control. Locoregional control was defined as complete and persistent regression of tumor within the irradiated field.
Oxygen Tension.
Oxygen tension was measured in the tumors before treatment (40
patients) and after about 2 weeks of radiotherapy (22 patients),
i.e., after a median radiation dose of 16 Gy was achieved by
external irradiation and before brachytherapy was initiated. General
anesthesia (Propofol i.v.) was used in nine patients subjected to a
single pO2 measurement, otherwise no anesthetic
was used. The anesthetic Propofol has no significant influence on body
temperature or tumor pO2 in cervical cancer
patients (18)
. The measurements were performed by use of
polarographic needle electrodes with a shaft diameter of 300 µm
(Eppendorf pO2 histograph 6650; Ref.
19
). The same electrode was generally used in the two
measurements of each patient. The electrode was moved automatically
through the tumor in two to six different tracks. The number of
pO2 readings in each tumor was 57–317 (median,
168). Five pO2 parameters were calculated for
each tumor: median pO2, fractions of
pO2 readings <2.5 mm Hg, 5 mm Hg, and 10 mm Hg
(HF2.5,
HF5, and
HF10), and the tumor subvolume with
pO2 readings <5 mm Hg
(HSV5, where
HSV5 = HF5
x tumor volume). Tumor volume was calculated as V =
/6 · a · b · c, where
a, b, and c are three orthogonal
diameters determined from MR images.
Biopsies.
A needle biopsy (1 x 18 mm) was taken from each measurement track
immediately after the pO2 electrode was withdrawn
from the track (19)
. Consequently, two to six biopsies
were achieved from each tumor before treatment (40 patients) and after
2 weeks of radiotherapy (22 patients). This procedure ensured that
possible heterogeneity in the histological parameters within the tumors
was taken into account. The biopsies were fixed in phosphate-buffered
4% paraformaldehyde, embedded in paraffin casts, and cut in the length
direction to 5-µm-thick sections. The sections were prepared as
described below and analyzed by one person (H. L.) in a light
microscope with an eyepiece grid for determination of vascular density,
cell density, and frequency of mitosis and apoptosis. The
reproducibility of the histological analyses was assessed by performing
repeated analyses of 10 sections. The first and second determinations
of the histological parameters were significantly correlated to each
other (P < 0.001), and there was no difference between
these two determinations, regardless of which parameter that was
considered (P < 0.85). All histological parameters
were therefore determined with satisfactory reproducibility.
Vascular Density.
Vascular density was determined in sections immunostained for factor
VIII-related antigen. A rabbit polyclonal antibody, Dako A0082 (Dako
Corp., Santa Barbara, CA), applied at a dilution of 1:500 at 20°C for
30 min, was used as primary antibody. Immunoperoxidase staining was
performed by using the Vectastain ABC peroxidase kit (Vector
Laboratories, Burlingame, CA) with goat-antirabbit IgG as biotinylated
secondary antibody and diaminobenzidine as chromogen. Brown-stained
endothelial cell clusters were identified as vessels. All sections of
each tumor were scanned at x100. The three areas (25
mm2) of highest vascular density were selected,
and all vessels within these areas were counted at x200. Vascular
density was calculated as number of vessels per
mm2 of tissue. The mean value based on the three
selected areas was used to represent vascular density of each tumor.
Cell Density.
Sections stained with H&E, using standard procedure, were used to
determine tumor cell density. Stroma, carcinoma tissue, and a
negligible amount of necrosis were seen in the sections
(19)
. Five fields, each generally including 50–150 cells,
were selected within the carcinoma tissue of each section. Carcinoma
cell nuclei were identified based on a blue color and a spherical
shape. All nuclei within the fields were counted at x400, and the
number of nuclei per mm2 of carcinoma tissue,
Dc, was determined. The area fraction
of carcinoma tissue, Fc, was
determined by point-counting at x100. Tumor cell density was defined
as number of carcinoma cell nuclei per mm2 of
tissue (including stroma and carcinoma tissue) and was calculated as
Dc x
Fc.
Frequency of Mitosis.
H&E-stained sections were used to determine mitotic frequency.
Carcinoma cell nuclei with morphological changes caused by chromosome
segregation were identified as mitotic cells. All mitotic cells were
counted by careful examination of the whole sections at x400. Mitotic
frequency (%) was calculated as the number of mitotic carcinoma cells
per number of all carcinoma cells.
Frequency of Apoptosis.
Apoptotic frequency was determined in sections stained by use of the
Apotag in situ apoptosis detection kit (Oncor,
Gaithersburg, MD). The staining was based on the TdT-mediated
dUTP-biotin nick end labeling method (20)
. Shortly, a
solution of 30% TdT was applied at 37°C for 60 min to link
dUTP-digoxigenin to the 3'-hydroxy ends of fragmented DNA.
Anti-digoxigenin peroxidase conjugate was applied for 30 min to detect
labeled nucleotides. Diaminobenzidine was used as chromogen. A biopsy
from a neoplastic lymph node of a patient with B-cell non-Hodgkin’s
lymphoma served as a positive control. Apoptotic frequency of this
lymph node was 
20%, as determined by flow cytometry earlier in our
institution. Negative controls received no TdT. To avoid erroneous
identification of apoptotic cells because of light staining of necrotic
cells, only brown-stained carcinoma nuclei with morphological
characteristics associated with apoptosis were identified as apoptotic
cells. Such characteristics include overall shrinkage, homogeneous dark
basophilia, a generally round or crescent shape, and a narrow empty
space often surrounding the nucleus. All apoptotic cells were counted
by careful examination of the whole sections at x400. Apoptotic
frequency (%) was calculated as number of apoptotic carcinoma cells
per number of all carcinoma cells.
Statistical Analysis.
The patients were divided into two groups based on high (above median)
and low (below median) value of the biological parameters. The control
probability was compared between the groups by actuarial analysis,
using a log-rank test in Kaplan-Meier estimates. Univariate and
multivariate Cox regression analyses of continuous data were used to
search for correlations between disease control and biological
parameters. A two-tailed t test or a Mann-Whitney rank sum
test was used, depending on whether the data were normally distributed,
to search for differences in biological parameters between two groups
of patients. A significance level of P = 0.05 was used
throughout.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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and
E). The failures are indicated by black symbols, using
overall survival (A), disease-free survival (C),
and locoregional control (E) as end point. The
HSV5 of all patients ranged from 3.3 to
184.5 cm3, with a median of 33.9
cm3. Failure occurred most often among patients
with a large HSV5. Patients with a
HSV5 above median had significantly lower
control probability than those with a HSV5
below median, regardless of whether overall survival, disease-free
survival, or locoregional control was used as an end point
(P < 0.001; Fig. 1, B, D, and F
). Moreover, there was a significant correlation between
survival and HSV5 (P <
0.001, overall and disease-free survival; P = 0.004,
locoregional control; Table 1
). The
HF5 was also related to control.
Patients with a HF5 above median had
significantly lower control probability than those with a
HF5 below median (P =
0.03, overall survival; P = 0.006, disease-free
survival; P = 0.02, locoregional control). The
correlation between HF5 and control
was significant when disease-free survival was used as end point
(P = 0.05) but on the borderline of significance when
overall survival (P = 0.08) or locoregional control
(P = 0.18) was considered in the analyses (Table 1)
.
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. Moreover, there was no difference in
control probability between patients with a high value and patients
with a low value of these parameters (Fig. 2)
. Pretreatment vascular density, cell
density, and frequency of mitosis and apoptosis were therefore probably
of minor importance for disease control compared with pretreatment
HSV5.
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; Table 1
). It was concluded that a parameter
was of minor importance for disease control compared with pretreatment
HSV5 if the correlation between control and
the parameter was weaker than the correlation between control and
pretreatment HSV5.
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.
Thus, HSV5 after 2 weeks of therapy showed
a weaker correlation to control (P = 0.13, overall
survival; P = 0.007, disease-free survival;
P = 0.33, locoregional control) than pretreatment
HSV5 (P = 0.02, overall
survival; P = 0.004, disease-free survival;
P = 0.06, locoregional control; Table 1
). Similar
results were achieved when the other pO2
parameters after 2 weeks of radiotherapy were considered
(HF5; Table 1
). The
pO2 after 2 weeks of radiotherapy was, therefore,
probably not as important as pretreatment
HSV5 for disease control.
Vascular density, cell density, and frequency of mitosis and apoptosis
after 2 weeks of therapy were not related to control either. Failure
occurred about equally as frequent, regardless of whether patients with
a high (above median) or a low (below median) value of these parameters
were considered (Fig. 4)
. All parameters
showed an impaired correlation to control (P > 0.4)
compared with pretreatment HSV5 (Table 1)
.
The vascular density, cell density, and frequency of mitosis and
apoptosis after 2 weeks of radiotherapy were, therefore, probably also
of minor importance for disease control compared with pretreatment
HSV5.
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for patients with
failure and patients with control, using overall survival
(A), disease-free survival (B), and locoregional
control (C) as end points. HSV5
decreased in 13 patients and increased in 9 patients. There were no
more failures among the patients with decreased
HSV5 than among those with increased
HSV5. The control probability was,
therefore, not increased for the former patients, although the HSV had
decreased. The magnitude of the changes in
HSV5 was not related to control either. The
changes, ranging from 38.1 cm3 to -91.2
cm3 for patients with failure and from + 22.9
cm3 to -55.3 cm3 for
patients with control (Fig. 5)
, were comparable with the pretreatment
HSV5, suggesting that an influence of the
changes on control should be detected, if present. The changes in
HSV5 showed a weaker correlation to disease
control (P = 0.09, overall survival; P = 0.24, disease-free survival; P = 0.09, locoregional
control) than the pretreatment HSV5 (Table 1)
. Thus, the changes in HSV5 during 2
weeks of radiotherapy were probably not as important as pretreatment
HSV5 for disease control. Analyses of
changes in the other pO2 parameters showed
similar results (HF5; Table 1
).
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shows changes in vascular density,
cell density, and frequency of mitosis and apoptosis during 2 weeks of
therapy. The cell density decreased significantly, both for patients
with failure and patients with control (P < 0.001;
Fig. 6B
), reflecting a marked treatment effect on the
tumors. The two groups of patients had also a significant increase in
apoptotic frequency (P = 0.03; Fig. 6D
).
Vascular density and mitotic frequency showed no changes during
therapy. The changes in the histological parameters showed an impaired
correlation to disease control (P > 0.2) compared with
pretreatment HSV5 (Table 1)
. The changes in
these parameters were, therefore, probably also of minor importance for
disease control compared with the HSV5.
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).
Analysis including stage, volume, and histological parameters
identified volume as an independent parameter (P =
0.0002, overall survival; P < 0.0001, disease-free
survival; P = 0.004, locoregional control; Table 2
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(a) The analyses may fail to identify true differences in control probability between patient groups (type I error). The aim of our work was to find the parameter most important for control. Because significant results were achieved when one of the parameters, pretreatment pO2, was considered in the analyses, the number of patients was large enough for our purpose. Although inclusion of more patients might lead to strong correlations between disease control and some of the other parameters also, the specificity of these parameters in prediction of control will probably not be as high as for pO2.
(b) Differences in control probability between patient groups may be erroneously identified (type II error). We found that patients with a high pretreatment pO2 had a higher control probability than patients with low pretreatment pO2, regardless of whether all patients or the subgroup of 22 patients was considered. Others have reported similar differences in studies based on a larger number of patients (14) , suggesting that our analyses identified true differences in control probability. Moreover, conclusions were based on the subgroup of 22 patients, assuming that the subgroup was representative of the whole group of patients. This assumption was probably fulfilled, because patient age, stage of disease, tumor volume, follow-up time, and biological parameters were about the same for the subgroup and the whole group of patients. It was, therefore, possible to draw conclusions based on the subgroup also.
The second measurement of biological parameters was performed after a treatment period of 2 weeks, i.e., before significant changes in tumor volume were expected. A tumor diameter of >2 cm is required for reliable pO2 measurements in cervical carcinomas with the Eppendorf pO2 Histograph (11) . Our choice of time point for the second measurement, therefore, ensured that no patients were rejected from this measurement because of comprehensive tumor shrinkage during treatment. Thus, in a previous study on cervix tumors, Fyles et al. (21) rejected >13% of the patients from the second pO2 measurement after a treatment period of 4 weeks because of insufficient tumor remaining. It should be emphasized, however, that the use of another time point for the second measurement may lead to relationships between control and the biological parameters that differ from those presented here, because considerable changes in the parameters may occur during the late phase of radiotherapy (22, 23, 24, 25) . Parameters measured during this phase or the changes in these parameters during that time may therefore be of significant importance for control of cervical cancer compared with pretreatment oxygenation.
Pretreatment pO2 showed a stronger correlation to disease control than the other biological parameters, indicating that the oxygenation is of major importance for control after radiotherapy of patients with cervical cancer. This hypothesis is supported by results from other studies (11 , 14 , 26) . The strong correlation between pretreatment pO2 and control suggests that the oxygenation influences rate-limiting steps of the events, leading to failure. Regional failure is the most common cause of death from this disease (27 , 28) . Failure can be seen as extensive tumor infiltration and lymphogeneous spread throughout and regionally beyond the pelvis, leading to obstruction of the ureters, loss of renal function, and uremia. The main factors determining control after radiotherapy are, therefore, probably incidence of lymphogeneous metastases and tumor radioresistance (29, 30, 31) . Hypoxia may induce genetic instability of tumors, leading to increased metastatic potential (32 , 33) . Our recent clinical studies showing that high incidence of lymph node metastases at the time of diagnosis is related to high lactate level and low oxygenation of the primary cervix tumor, suggest a significant influence of oxygenation on the metastatic process (34 , 35) . Increased radioresistance of hypoxic tumors is, however, also a probable factor contributing to the impaired disease control of patients with poorly oxygenated cervix tumors (36) .
Although several studies indicate that pretreatment oxygenation is of major importance for disease control of cervical cancer, it is not clear whether the oxygenation during radiotherapy is important (21 , 37) . Our results suggest that the oxygenation after 2 weeks of radiotherapy and the changes in the parameter during this time are of minor importance for disease control compared with the pretreatment oxygenation. Similarly, Fyles et al. (21) reported impaired correlation between oxygenation and survival of patients with cervical cancer when hypoxic fraction after 4 weeks of radiotherapy rather than pretreatment hypoxic fraction was considered. There may be several reasons for these observations:
(a) Metastasis formation often occurs during an early phase of the disease, i.e., before treatment is started, and may therefore depend primarily on pretreatment oxygenation and not on the oxygenation during therapy. Thus, lymphogeneous spread of cervical cancer is common, even in early-stage carcinomas, and occurs in 25–50% of patients with stages Ib and II (28 , 38) .
(b) The hypoxia-induced radioresistance, depending on the oxygenation and clonogenicity at each time of irradiation, may not be very well reflected by the oxygenation after a certain time of radiotherapy. Thus, the oxygenation may fluctuate between high and low values during therapy, depending on changes in biological parameters important for the oxygen consumption and supply of the tumor (39 , 40) . An increase in oxygenation during the early phase of radiotherapy may be followed by a decrease during a later phase and vice versa. Pretreatment oxygenation may be a better indicator of the radioresistance because of a higher clonogenicity before treatment.
Tumor vascular density, cell density, and frequency of mitosis and apoptosis were also of minor importance for disease control compared with pretreatment oxygenation, regardless of whether the pretreatment parameters, the parameters after 2 weeks of radiotherapy, or the changes in the parameters during this time were considered. In accordance with our data, Hawighorst et al. (17) found no correlation between pretreatment vascular density and control in a study of 37 patients with cervical cancer. Moreover, pretreatment apoptotic frequency showed only a weak correlation to control in a study of 44 patients with this disease (8) . It is possible that studies based on more patients than included here would show significant correlations between control and some of the parameters other than pretreatment oxygenation. Thus, pretreatment vascular density has been found recently to influence control of cervical cancer in studies of >100 patients (15 , 41) . However, the need for a large number of patients to achieve significant results suggests that these parameters are not as important as pretreatment oxygenation for disease control.
The present results indicate that pretreatment oxygenation is more important for disease control of cervical cancer than the oxygenation after 2 weeks of radiotherapy or the changes in the parameter during this time. Moreover, vascular density, cell density, and frequency of mitosis and apoptosis before treatment or after 2 weeks of therapy are probably not as important as pretreatment oxygenation for control as well. Significant correlations between disease control and the oxygenation were achieved for 40 patients, suggesting a high specificity of this parameter in prediction of control. Selection of patients to adjuvant treatments should therefore be based on pretreatment measurements of tumor oxygenation. Development of efficient strategies for influencing the control probability by changing tumor oxygenation probably necessitates knowledge of whether increased metastatic potential or increased radioresistance is the major cause of the impaired disease control of patient with hypoxic cervix tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Support was received from The Norwegian Cancer
Society and The Bothner Foundation for Cancer Research. 
2 To whom requests for reprints should be
addressed, at Department of Biophysics, The Norwegian Radium Hospital,
Montebello, 0310 Oslo 3, Norway. Phone: 47-2293-4258; Fax:
47-2293-4270; E-mail: heidi.lyng{at}labmed.uio.no
3 The abbreviations used are: MR, magnetic
resonance; Tdt, terminal deoxynucleotidyl transferase; HSV, hypoxic
subvolume.
Received 4/ 1/99; revised 10/15/99; accepted 12/21/99.
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. Oxygen tension and prediction of the radiation response. Polarographic study in human breast cancer. Neoplasma, 29: 669-674, 1982.[Medline]
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) or failure (•) are indicated, using
overall survival (A), disease-free survival
(C), and locoregional control (E) as end
points; each symbol represents one patient; the median
value of the HSV is marked (····). B, D, and
F, patients with pretreatment HSV below the median value
(
) and above the median value (
) are compared, using overall
survival (B), disease-free survival (D),
and locoregional control (F) as end points; each
symbol represents a censored observation.
) are compared, using disease-free survival
as an end point; each symbol represents a censored
observation.
